PROJECT SUMMARY Diabetes is a growing pandemic that is characterized by insufficiency of insulin, a key hormone of glucose maintenance. Ca2+ influx through voltage-gated calcium channels (VGCCs) is necessary for glucose-stimulated insulin secretion in pancreatic ?-cells (P?Cs), and has also been implicated in maintaining P?C identity, an important regulatory point in diabetes progression. VGCCs are thus a potential locus for both P?C-dependent pathophysiology and therapy. These channels are multi-subunit complexes comprised minimally of pore-forming ?1 subunits assembled with auxiliary (?, ?2?,and ?) proteins. In heterologous cells, CaV? subunits (?1-?4) are powerful regulators of VGCCs by controlling ?1 subunit trafficking and tuning channel gating. A central unresolved question is: how does Ca2+ influx via VGCCs give rise to divergent functions in P?Cs such as insulin secretion and excitation-transcription coupling? It is likely that differential sorting of VGCCs into spatially distinct macromolecular complexes is a key underlying principle that enables such functional diversification of VGCC Ca2+ signals. P?Cs express multiple CaV?1 and ? subunits? the precise functional roles of distinct CaV?s in P?C physiology and pathophysiology are unclear. Nevertheless, CaV?s are downregulated in rodent models of diabetes, and knockout models suggest they play a role in glucose homeostasis. I hypothesize that in P?Cs distinct CaV?s are instrumental in organizing VGCCs into discrete macromolecular complexes with specialized functions. A significant barrier in our capacity to rigorously assess the functional roles of CaV? molecular diversity in excitable cells, including P?Cs, is the inability to inhibit VGCCs based on the identity of their resident CaV?. Here, I propose to develop novel genetically-encoded CaV channel blockers that enable inhibition of CaV?-specific VGCC complexes, and apply them to decipher signaling functions of CaV?s in P?Cs. The approach exploits a bioengineering method to generate genetically-encoded VGCC inhibitors termed Channel Inactivation by Membrane-tethering of an Associated Protein (ChIMP) pioneered by our lab. In this proposal, I combine development of Cav? isoform-selective nanobodies with molecular biology, electrophysiology, flow cytometry, fluorescence resonance energy transfer (FRET), ion channel engineering, and biochemistry to address two specific aims. First, I will develop and engineer nanobodies to selectively inhibit VGCCs on the basis of their resident CaV?. In my second aim I will elucidate the functions of CaV? specific VGCC complexes in pancreatic ?-cells.